ATP-Coated Dual-Functionalized Titanium(IV) IMAC Material for Simultaneous Enrichment and Separation of Glycopeptides and Phosphopeptides.

Protein glycosylation and phosphorylation are two of the most common post-translational modifications (PTMs), which play an important role in many biological processes. However, low abundance and poor ionization efficiency of phosphopeptides and glycopeptides make direct MS analysis challenging. In this study, we developed a hydrophilicity-enhanced bifunctional Ti-IMAC (IMAC: immobilized metal affinity chromatography) material with grafted adenosine triphosphate (denoted as epoxy-ATP-Ti4+) to enable simultaneous enrichment and separation of common N-glycopeptides, phosphopeptides, and M6P glycopeptides from tissue/cells. The enrichment was achieved through a dual-mode mechanism based on the electrostatic and hydrophilic properties of the material. The epoxy-ATP-Ti4+ IMAC material was prepared from epoxy-functionalized silica particles via a convenient two-step process. The ATP molecule provided strong and active phosphate sites for binding phosphopeptides in the conventional IMAC mode and also contributed significantly to the hydrophilicity, which permitted the enrichment of glycopeptides via hydrophilic interaction chromatography. The two modes could be implemented simultaneously, allowing glycopeptides and phosphopeptides to be collected sequentially in a single experiment from the same sample. In addition to standard protein samples, the material was further applied to glycopeptide and phosphopeptide enrichment and characterization from HeLa cell digests and mouse lung tissue samples. In total, 2928 glycopeptides and 3051 phosphopeptides were identified from the mouse lung tissue sample, supporting the utility of this material for large-scale PTM analysis of complex biological samples. Overall, the newly developed epoxy-ATP-Ti4+ IMAC material and associated fractionation method enable simple and effective enrichment and separation of glycopeptides and phosphopeptides, offering a useful tool to study potential crosstalk between these two important PTMs in biological systems. The MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD029775.

DiLeu Isobaric Labeling Coupled with Limited Proteolysis Mass Spectrometry for High-Throughput Profiling of Protein Structural Changes in Alzheimer's Disease.

High-throughput quantitative analysis of protein conformational changes has a profound impact on our understanding of the pathological mechanisms of Alzheimer's disease (AD). To establish an effective workflow enabling quantitative analysis of changes in protein conformation within multiple samples simultaneously, here we report the combination of N,N-dimethyl leucine (DiLeu) isobaric tag labeling with limited proteolysis mass spectrometry (DiLeu-LiP-MS) for high-throughput structural protein quantitation in serum samples collected from AD patients and control donors. Twenty-three proteins were discovered to undergo structural changes, mapping to 35 unique conformotypic peptides with significant changes between the AD group and the control group. Seven out of 23 proteins, including CO3, CO9, C4BPA, APOA1, APOA4, C1R, and APOA, exhibited a potential correlation with AD. Moreover, we found that complement proteins (e.g., CO3, CO9, and C4BPA) related to AD exhibited elevated levels in the AD group compared to those in the control group. These results provide evidence that the established DiLeu-LiP-MS method can be used for high-throughput structural protein quantitation, which also showed great potential in achieving large-scale and in-depth quantitative analysis of protein conformational changes in other biological systems.

Selective Enrichment of Sialylglycopeptides Enabled by Click Chemistry and Dynamic Covalent Exchange.

Despite the important roles of protein sialylation in biological processes such as cellular interaction and cancer progression, simple and effective methods for the analysis of intact sialylglycopeptides (SGPs) are still limited. Analyses of low-abundance SGPs typically require efficient enrichment prior to comprehensive liquid chromatography-mass spectrometry (LC-MS)-based analysis. Here, a novel workflow combining mild periodate oxidation, hydrazide chemistry, copper-catalyzed azide/alkyne cycloaddition (CuAAC) click chemistry, and dynamic covalent exchange has been developed for selective enrichment of SGPs. The intact SGPs could be separated easily from protein tryptic digests, and the signature ions were produced during LC-MS/MS for unambiguous identification. The structure of the signature ions and corresponding dynamic covalent exchange were confirmed by using an isotopic reagent. Under the optimized condition, over 70% enrichment efficiency of SGPs was achieved using bovine fetuin digests, and the method was successfully applied to complex biological samples, such as a mouse lung tissue extract. The high enrichment efficiency, good reproducibility, and easily adopted procedure without the need to generate specialized materials make this method a promising tool for broad applications in SGP analysis.

Enabling Global Analysis of Protein Citrullination via Biotin Thiol Tag-Assisted Mass Spectrometry.

Citrullination is a key post-translational modification (PTM) that affects protein structures and functions. Although it has been linked to various biological processes and disease pathogenesis, the underlying mechanism remains poorly understood due to a lack of effective tools to enrich, detect, and localize this PTM. Herein, we report the design and development of a biotin thiol tag that enables derivatization, enrichment, and confident identification of citrullination via mass spectrometry. We perform global mapping of the citrullination proteome of mouse tissues. In total, we identify 691 citrullination sites from 432 proteins which represents the largest data set to date. We discover novel distribution and functions of this PTM. This study depicts a landscape of protein citrullination and lays the foundation for further deciphering their physiological and pathological roles.

On-tissue amidation of sialic acid with aniline for sensitive imaging of sialylated N-glycans from FFPE tissue sections via MALDI mass spectrometry.

Spatial visualization of glycans within clinical tissue samples is critical for discovery of disease-relevant glycan dysregulations. Herein, we develop an on-tissue derivatization strategy for sensitive spatial visualization of N-glycans from formalin-fixed paraffin-embedded (FFPE) tissue sections, based on amidation of sialic acid residues with aniline. The sialylated N-glycans were stabilized and given enhanced signal intensity owing to selective capping of a phenyl group to the sialic acid residue after aniline labeling. Proof-of-concept experiments, including determinations of sialylglycopeptide and N-glycans enzymatically released from glycoproteins, were performed. Further, mass spectrometry (MS) imaging of N-glycans on human laryngeal cancer FFPE tissue sections was conducted via matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), based on our strategy for on-tissue amidation of sialylated N-glycans. We obtained higher sialylated N-glycan coverages for both the glycoproteins and cancer tissue samples, demonstrating that the detection sensitivity for sialylated N-glycans is notably improved by amidation derivatization. We also characterized N-glycan heterogeneity across the human laryngeal cancer tissue section, showing N-glycan dysregulation in the tumor region.

Comparative transcriptomic analysis of THP-1-derived macrophages infected with Mycobacterium tuberculosis H37Rv, H37Ra and BCG.

Tuberculosis (TB) remains a worldwide healthcare concern, and the exploration of the host-pathogen interaction is essential to develop therapeutic modalities and strategies to control Mycobacterium tuberculosis (M.tb). In this study, RNA sequencing (transcriptome sequencing) was employed to investigate the global transcriptome changes in the macrophages during the different strains of M.tb infection. THP-1 cells derived from macrophages were exposed to the virulent M.tb strain H37Rv (Rv) or the avirulent M.tb strain H37Ra (Ra), and the M.tb BCG vaccine strain was used as a control. The cDNA libraries were prepared from M.tb-infected macrophages and then sequenced. To assess the transcriptional differences between the expressed genes, the bioinformatics analysis was performed using a standard pipeline of quality control, reference mapping, differential expression analysis, protein-protein interaction (PPI) networks, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Q-PCR and Western blot assays were also performed to validate the data. Our findings indicated that, when compared to BCG or M.tb H37Ra infection, the transcriptome analysis identified 66 differentially expressed genes in the M.tb H37Rv-infected macrophages, out of which 36 genes were up-regulated, and 30 genes were down-regulated. The up-regulated genes were associated with immune response regulation, chemokine secretion, and leucocyte chemotaxis. In contrast, the down-regulated genes were associated with amino acid biosynthetic and energy metabolism, connective tissue development and extracellular matrix organization. The Q-PCR and Western blot assays confirmed increased expression of pro-inflammatory factors, altered energy metabolic processes, enhanced activation of pro-inflammatory signalling pathways and increased pyroptosis in H37Rv-infected macrophage. Overall, our RNA sequencing-based transcriptome study successfully identified a comprehensive, in-depth gene expression/regulation profile in M.tb-infected macrophages. The results demonstrated that virulent M.tb strain H37Rv infection triggers a more severe inflammatory immune response associated with increased tissue damage, which helps in understanding the host-pathogen interaction dynamics and pathogenesis features in different strains of M.tb infection.

Dual-Functional Ti(IV)-IMAC Material Enables Simultaneous Enrichment and Separation of Diverse Glycopeptides and Phosphopeptides.

Simultaneous enrichment and fractionation of diverse proteins/peptides possessing different post-translational modifications (PTMs) from the same biological samples is highly desirable to reduce sample consumption, avoid complicated sample processing, and enable studies of potential crosstalks between different PTMs. In this work, we report a new approach to enable simultaneous enrichment and separation of glycopeptides, phosphopeptides, and mannose-6-phosphate (M6P) glycopeptides by using a dual-functional Ti(IV)-IMAC material. Moreover, we also made the separation of neutral and sialyl glycopeptides and mono- and multi-phosphopeptides possible by performing different elution processes according to the differences in their electrostatic or hydrophilic properties. These separations are effective and efficient to eliminate the signal suppression from neutral glycopeptides for sialyl glycopeptide detection, allowing separation of mono-phosphopeptides from multi-phosphopeptides, as well as detection of M6P glycopeptides that are free from the abovementioned modifications. This new strategy significantly improves the coverage and identification numbers of glycopeptides, phosphopeptides, and M6P glycopeptides by 1.9, 2.3, and 4.3-fold compared with the conventional method, respectively. This is the first report on simultaneous enrichment and separation of neutral and sialyl glycopeptides, mono- and multi-phosphopeptides, and M6P glycopeptides via dual-functional Ti(IV)- IMAC, revealing novel insights into potential crosstalk among these important PTMs.

High-resolution magnetic resonance and mass spectrometry imaging of the human larynx.

High-resolution, noninvasive and nondestructive imaging of the subepithelial structures of the larynx would enhance microanatomic tissue assessment and clinical decision making; similarly, in situ molecular profiling of laryngeal tissue would enhance biomarker discovery and pathology readout. Towards these goals, we assessed the capabilities of high-resolution magnetic resonance imaging (MRI) and matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) imaging of rarely reported paediatric and adult cadaveric larynges that contained pathologies. The donors were a 13-month-old male, a 10-year-old female with an infraglottic mucus retention cyst and a 74-year-old female with advanced polypoid degeneration and a mucus retention cyst. MR and molecular imaging data were corroborated using whole-organ histology. Our MR protocols imaged the larynges at 45-117 µm2 in-plane resolution and capably resolved microanatomic structures that have not been previously reported radiographically-such as the vocal fold superficial lamina propria, vocal ligament and macula flavae; age-related tissue features-such as intramuscular fat deposition and cartilage ossification; and the lesions. Diffusion tensor imaging characterised differences in water diffusivity, primary tissue fibre orientation, and fractional anisotropy between the intrinsic laryngeal muscles, mucosae and lesions. MALDI-MS imaging revealed peptide signatures and putative protein assignments for the polypoid degeneration lesion and the N-glycan constituents of one mucus retention cyst. These imaging approaches have immediate application in experimental research and, with ongoing technology development, potential for future clinical application.

On-Tissue Derivatization with Girard's Reagent P Enhances N-Glycan Signals for Formalin-Fixed Paraffin-Embedded Tissue Sections in MALDI Mass Spectrometry Imaging.

Glycosylation is a major protein post-translational modification whose dysregulation has been associated with many diseases. Herein, an on-tissue chemical derivatization strategy based on positively charged hydrazine reagent (Girard's reagent P) coupled with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed for analysis of N-glycans from FFPE treated tissue sections. The performance of the proposed approach was evaluated by analysis of monosaccharides, oligosaccharides, N-glycans released from glycoproteins, as well as MS imaging of N-glycans from human cancer tissue sections. The results demonstrated that the signal-to-noise ratios for target saccharides were notably improved after chemical derivatization, in which signals were enhanced by 230-fold for glucose and over 28-fold for maltooctaose. Improved glycome coverage was obtained for N-glycans derived from glycoproteins and tissue samples after chemical derivatization. Furthermore, on-tissue derivatization was applied for MALDI-MSI of N-glycans from human laryngeal cancer and ovarian cancer tissues. Differentially expressed N-glycans among the tumor region, adjacent normal tissue region, and tumor proximal collagen stroma region were imaged, revealing that high-mannose type N-glycans were predominantly expressed in the tumor region. Overall, our results indicate that the on-tissue labeling strategy coupled with MALDI-MSI shows great potential to spatially characterize N-glycan expression within heterogeneous tissue samples with enhanced sensitivity. This study provides a promising approach to better understand the pathogenesis of cancer related aberrant glycosylation, which is beneficial to the design of improved clinical diagnosis and therapeutic strategies.

Multifunctional Transferrin Encapsulated GdF3 Nanoparticles for Sentinel Lymph Node and Tumor Imaging.

The transferrin encapsulated GdF3 nanoparticles have been fabricated via biomineralization method. The obtained GdF3@Tf NPs show an attractive T2MRI and CT enhancement effect. Furthermore, PET and NIR imaging capacity are integrated into nanoparticles through conjugating with radionuclide 64Cu and fluorescent dye Cy7. 64Cu-GdF3@Tf-Cy7 NPs are developed and applied in small animal multimodal imaging in vivo. Compared with the previous multimodal imaging agents, 64Cu-GdF3@Tf-Cy7 NPs enable not only precise sentinel lymph node (SLN) identification, but specific imaging for transferrin receptor overexpressed colorectal tumor in vivo. The results reveal that 64Cu-GdF3@Tf-Cy7 NPs are potential and efficient multimodal imaging agents for SLN and tumor preclinical imaging.

Combined tests with Xpert MTB/RIF assay with bronchoalveolar lavage fluid increasing the diagnostic performance of smear-negative pulmonary tuberculosis in Eastern China.

Tuberculosis (TB) remains a global public health threat. Misdiagnosis and delayed therapy of sputum smear-negative TB can affect the treatment outcomes and promote pathogen transmission. The application of Xpert MTB/RIF assay in bronchoalveolar lavage fluid (BALF) has been recommended but needs clinical evidence. We carried out a prospective study in the Nanjing Public Health Medical Center from September 2018 to August 2019. Pulmonary tuberculosis (PTB) patients were enrolled in the study if they had negative results of sputum smear. We compared the performance of Xpert MTB/RIF assay in sputum and BALF using sputum culture as the reference. In addition to this, we applied parallel tests using sputum culture, sputum-based Xpert MTB/RIF assay and BALF-based Xpert MTB/RIF assay to jointly detect smear-negative PTB using clinical diagnosis as the reference. With mycobacterial culture as the reference standard, Xpert MTB/RIF of BALF showed a higher sensitivity (14/16, 87.5%), but a relatively lower specificity (57/92, 62.0%). Xpert MTB/RIF of sputum showed relatively lower sensitivity (6/10, 60.0%) and higher specificity (63/88, 71.6%). Compared with sputum culture, Xpert MTB /RIF assay reduced the median detection time of MTB from 30 to 0 days, which significantly shortened the diagnosis time of the smear-negative TB patients. Among the combined detections, the positive detection proportion was improved with significant differences comparing with sputum culture only, from 11.1% (10/90) to 46.7% (42/90) (P < 0.05). Our study showed Xpert MTB/RIF in BALF had a better performance in detecting MTB of smear-negative patients.

Mass Spectrometric Imaging Reveals Temporal and Spatial Dynamics of Bioactive Lipids in Arteries Undergoing Restenosis.

Restenosis, or renarrowing of the arterial lumen, is a common recurrent disease following balloon angioplasty and stenting treatments for cardiovascular disease. A major technical barrier for deciphering restenotic mechanisms is the dynamic, spatial profiling of bioactive lipids in the arterial wall, especially in small animals. Here, applying matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI), we conducted the first lipidomic study of temporal-spatial profiling in a small animal model of angioplasty-induced restenosis. Cross sections were collected 3, 7, and 14 days after balloon angioplasty of rat carotid arteries. MALDI-MSI analyses showed that diacylglycerols (DAGs), signaling lipids associated with restenosis, and lysophosphatidylcholines (LysoPCs), whose function was uncharacterized in restenosis, dramatically increased at postangioplasty day 7 and day 14 in the neointimal layer of balloon-injured arteries compared to uninjured controls. In contrast, sphingomyelins (SMs) did not increase, but rather decreased at day 3, day 7, and day 14 in injured arteries versus the uninjured control arteries. These results revealed previously unexplored distinct temporal-spatial lipid dynamics in the restenotic arterial wall. Additionally, we employed time-of-flight secondary ion mass spectrometry (TOF-SIMS) tandem MS imaging for both molecular identification and imaging at high spatial resolution. These imaging modalities provide powerful tools for unraveling novel mechanisms of restenosis involving lipids or small signaling molecules.

Dual-Functional Titanium(IV) Immobilized Metal Affinity Chromatography Approach for Enabling Large-Scale Profiling of Protein Mannose-6-Phosphate Glycosylation and Revealing Its Predominant Substrates.

Mannose-6-phosphate (M6P) glycosylation is an important post-translational modification (PTM) and plays a crucial role in transferring lysosomal hydrolases to lysosome, and is involved in several other biological processes. Aberrant M6P modifications have been implicated in lysosomal storage diseases and numerous other disorders including Alzheimer's disease and cancer. Research on profiling of intact M6P glycopeptides remains challenging due to its extremely low stoichiometry. Here we propose a dual-mode affinity approach to enrich M6P glycopeptides by dual-functional titanium(IV) immobilized metal affinity chromatography [Ti(IV)-IMAC] materials. In combination with state-of-the-art mass spectrometry and database search engine, we profiled 237 intact M6P glycopeptides corresponding to 81 M6P glycoproteins in five types of tissues in mouse, representing the first large-scale profiling of M6P glycosylation in mouse samples. The analysis of M6P glycoforms revealed the predominant glycan substrates of this PTM. Gene ontology analysis showed that overrepresented M6P glycoproteins were lysosomal-associated proteins. However, there were still substantial M6P glycoproteins that possessed different subcellular locations and molecular functions. Deep mining of their roles implicated in lysosomal and nonlysosomal function can provide new insights into functional roles of this important yet poorly studied modification.

Mass Spectrometry Imaging of N-Glycans from Formalin-Fixed Paraffin-Embedded Tissue Sections Using a Novel Subatmospheric Pressure Ionization Source.

N-linked glycosylation, featuring various glycoforms, is one of the most common and complex protein post-translational modifications (PTMs) controlling protein structures and biological functions. It has been revealed that abnormal changes of protein N-glycosylation patterns are associated with many diseases. Hence, unraveling the disease-related alteration of glycosylation, especially the glycoforms, is crucial and beneficial to improving our understanding about the pathogenic mechanisms of various diseases. In past decades, given the capability of in situ mapping of biomolecules and their region-specific localizations, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been widely applied to the discovery of potential biomarkers for many diseases. In this study, we coupled a novel subatmospheric pressure (SubAP)/MALDI source with a Q Exactive HF hybrid quadrupole-orbitrap mass spectrometer for in situ imaging of N-linked glycans from formalin-fixed paraffin-embedded (FFPE) tissue sections. The utility of this new platform for N-glycan imaging analysis was demonstrated with a variety of FFPE tissue sections. A total of 55 N-glycans were successfully characterized and visualized from a FFPE mouse brain section. Furthermore, 29 N-glycans with different spatial distribution patterns could be identified from a FFPE mouse ovarian cancer tissue section. High-mannose N-glycans exhibited elevated expression levels in the tumor region, indicating the potential association of this type of N-glycans with tumor progression.

Characterization of non-tuberculous mycobacterial pulmonary disease in Nanjing district of China.

BACKGROUND: Environmental bacteria, nontuberculous mycobacteria (NTM), are recognized as one of the major human infection pathogens. NTM are prone to be mistaken as multidrug-resistant Mycobacterium tuberculosis and challenge our fight against TB. In addition, treatment of NTM per se is intractable. Remarkably, the distribution of NTM pathogenic species is geographically specific. Thus, it is very important to summarize the prevalent features and clinical symptoms of NTM pulmonary disease. However, In Nanjing district, southeast China, there is no such a report. METHODS: Through investigating electronic medical records and analyzing data of clinical examination system (Lis), we retrospectively summarized the NTM species from 6012 clinical isolates from May 2017 to August 2018, and analyzed the association between NTM species and clinical symptoms. RESULTS: Of 6012 clinical specimens, 1461 (24.3%) could grow in the MGIT 960 broth. Among these positive isolates, 1213 (83%) were M. tuberculosis, 22 (1.5%) were M. bovis, and 226 (15.5%) were NTM. After deducting redundancy, those NTM specimens were confirmed from 154 patients, among which, 87 (56.5%) patients met the full ATS/IDSA NTM disease criteria. The most common etiologic agent was M. intracellulare (70.1%). NTM infection was associated with age, based on which 68.6% male patients and 77.8% female patients were over 50 years old. The older patients were more likely to have hemoptysis, but the younger patients were more likely to manifest chest congestion. Male patients were more likely to have shortness of breath and females were more likely to have hemoptysis. The most common radiographic presentation of NTM pulmonary disease was bronchiectasis, accounting for 39.1%. Remarkably, multiple and thin-walled cavities were outstanding. The most frequent comorbidity of NTM disease was previous tuberculosis (64%), followed by clinical bronchiectasis (19.5%), HIV (19.5%), and 6.9% chronic obstructive pulmonary disease (COPD). There was no association between NTM species and clinical symptoms. CONCLUSION: This study retrospectively investigated the prevalence of NTM pulmonary disease in Nanjing district, southeast China. Similar to Beijing area, north China, M. intracellulare was the major pathogenic NTM species. Clinical symptoms of the disease were not species-specific. Previous TB and HIV infection immensely enhanced risk of NTM disease.

Pretargeted Immuno-PET Based on Bioorthogonal Chemistry for Imaging EGFR Positive Colorectal Cancer.

Pretargeted immuno-PET imaging based on the bioorthogonal chemistry between 18F-labeled Reppe anhydride derivatives and tetrazine conjugates of the EGFR-specific monoclonal antibodies cetuximab and panitumumab was performed. This pretargeting approach yielded high target-to-nontarget ratios. Furthermore, due to the fast clearance rate of the PET probe, the overall radiation burden to nontarget tissues was also substantially decreased.

Targeted Mass Spectrometry Approach Enabled Discovery of O-Glycosylated Insulin and Related Signaling Peptides in Mouse and Human Pancreatic Islets.

O-Linked glycosylation often involves the covalent attachment of sugar moieties to the hydroxyl group of serine or threonine on proteins/peptides. Despite growing interest in glycoproteins, little attention has been directed to glycosylated signaling peptides, largely due to lack of enabling analytical tools. Here we explore the occurrence of naturally O-linked glycosylation on the signaling peptides extracted from mouse and human pancreatic islets using mass spectrometry (MS). A novel targeted MS-based method is developed to increase the likelihood of capturing these modified signaling peptides and to provide improved sequence coverage and accurate glycosite localization, enabling the first large-scale discovery of O-glycosylation on signaling peptides. Several glycosylated signaling peptides with multiple glycoforms are identified, including the first report of glycosylated insulin-B chain and insulin-C peptide and BigLEN. This discovery may reveal potential novel functions as glycosylation could influence their conformation and biostability. Given the importance of insulin and its related peptide hormones and previous studies of glycosylated insulin analogues, this natural glycosylation may provide important insights into diabetes research and therapeutic treatments.

Local CXCR4 Upregulation in the Injured Arterial Wall Contributes to Intimal Hyperplasia.

CXCR4 is a stem/progenitor cell surface receptor specific for the cytokine stromal cell-derived factor-1 (SDF-1α). There is evidence that bone marrow-derived CXCR4-expressing cells contribute to intimal hyperplasia (IH) by homing to the arterial subintima which is enriched with SDF-1α. We have previously found that transforming growth factor-ß (TGFß) and its signaling protein Smad3 are both upregulated following arterial injury and that TGFß/Smad3 enhances the expression of CXCR4 in vascular smooth muscle cells (SMCs). It remains unknown, however, whether locally induced CXCR4 expression in SM22 expressing vascular SMCs plays a role in neointima formation. Here, we investigated whether elevated TGFß/Smad3 signaling leads to the induction of CXCR4 expression locally in the injured arterial wall, thereby contributing to IH. We found prominent CXCR4 upregulation (mRNA, 60-fold; protein, 4-fold) in TGFß-treated, Smad3-expressing SMCs. Chromatin immunoprecipitation assays revealed a specific association of the transcription factor Smad3 with the CXCR4 promoter. TGFß/Smad3 treatment also markedly enhanced SDF-1α-induced ERK1/2 phosphorylation as well as SMC migration in a CXCR4-dependent manner. Adenoviral expression of Smad3 in balloon-injured rat carotid arteries increased local CXCR4 levels and enhanced IH, whereas SMC-specific depletion of CXCR4 in the wire-injured mouse femoral arterial wall produced a 60% reduction in IH. Our results provide the first evidence that upregulation of TGFß/Smad3 in injured arteries induces local SMC CXCR4 expression and cell migration, and consequently IH. The Smad3/CXCR4 pathway may provide a potential target for therapeutic interventions to prevent restenosis. Stem Cells 2016;34:2744-2757.

Unimolecular Micelle-Based Hybrid System for Perivascular Drug Delivery Produces Long-Term Efficacy for Neointima Attenuation in Rats.

At present, there are no clinical options for preventing neointima-caused (re)stenosis after open surgery such as bypass surgery for treating flow-limiting vascular disease. Perivascular drug delivery is a promising strategy, but in translational research, it remains a major challenge to achieve long-term (e.g., > 3 months) anti(re)stenotic efficacy. In this study, we engineered a unique drug delivery system consisting of durable unimolecular micelles, formed by single multiarm star amphiphilic block copolymers with only covalent bonds, and a thermosensitive hydrogel formed by a poly(lactide-co-glycolide)-poly(ethylene glycol)-poly(lactide-co-glycolide) triblock copolymer (abbreviated as triblock gel) that is stable for about 4 weeks in vitro. The drug-containing unimolecular micelles (UMs) suspended in Triblock gel were able to sustain rapamycin release for over 4 months. Remarkably, even 3 months after perivascular application of the rapamycin-loaded micelles in Triblock gel in the rat model, the intimal/medial area ratio (a restenosis measure) was still 80% inhibited compared to the control treated with empty micelle/gel (no drug). This could not be achieved by applying rapamycin in Triblock gel alone, which reduced the intimal/medial ratio only by 27%. In summary, we created a new UM/Triblock gel hybrid system for perivascular drug delivery, which produced a rare feat of 3-month restenosis inhibition in animal tests. This system exhibits a real potential for further translation into an anti(re)stenotic application with open surgery.

Evaluation and Application of Dimethylated Amino Acids as Isobaric Tags for Quantitative Proteomics of the TGF-ß/Smad3 Signaling Pathway.

Isobaric labeling has become a widespread tool for quantitative proteomic studies. Here, we report the development and evaluation of several dimethylated amino acids as novel isobaric tags for quantitative proteomics. Four-plex dimethylated alanine (DiAla), valine (DiVal), and leucine (DiLeu) have been synthesized, sharing common features of peptide tagging and reporter ion production. DiAla and DiLeu are shown to achieve complete labeling. These two tags' impacts on peptide fragmentation and quantitation are further evaluated using HEK293 cell lysate. DiAla labeling generates more abundant backbone fragmentation whereas DiLeu labeling produces more intense reporter ions. Nonetheless, both tags enable accurate quantitative analysis of HEK293 cell proteomes. DiAla and DiLeu tags are then applied to study the TGF-ß/Smad3 pathway with four differentially treated mouse vascular smooth muscle (MOVAS) cells. Our MS data reveal proteome-wide changes of AdSmad3 as compared to the GFP control, consistent with previous findings of causing smooth muscle cell (SMC) dedifferentiation.1 Additionally, the other two novel mutations on the hub protein Smad3, Y226A, and D408H, show compromised TGF-ß/Smad3-dependent gene transcription and reversed phenotypic switch. These results are further corroborated with Western blotting and demonstrate that the novel DiAla and DiLeu isobaric tagging reagents provide useful tools for multiplex quantitative proteomics.